Methods of manufacture of therapeutic products comprising vitalized placental dispersions

ABSTRACT

This invention provides a fluid therapeutic placental product comprising placental cells and a placental dispersion comprising placental factors. The placental cells and the placental dispersion are derived from placental tissue. A placental tissue can optionally be an amnion, chorion, or a trophoblast-depleted chorion. The placental product of the present invention is useful in treating a patient with a tissue injury (e.g. wound or burn) by applying the placental product to the injury. Similar application is useful with ligament and tendon repair and for engraftment procedures such as bone engraftment.

RELATED APPLICATIONS

This application claims priority to:

-   -   U.S. Provisional Application Ser. No. 61/338,464 entitled        “Selectively Immunodepleted Chorionic Membranes”, filed on Feb.        18, 2010 bearing Docket No. 22924US01,    -   U.S. Provisional Application Ser. No. 61/338,489 entitled        “Selectively Immunodepleted Amniotic Membranes”, filed on Feb.        18, 2010 bearing Docket No. 22925US01, and    -   U.S. Provisional Application Ser. No. 61/369,562 entitled        “Therapeutic Products Comprising Vitalized Placental Dispersions        filed on Jul. 30, 2010 bearing Docket No 23498US01, the contents        of which are hereby incorporated by reference in their        entireties.

This application is being co-filed on Feb. 18, 2011 with, andincorporates by reference, applications entitled:

-   -   “Immunocompatible Chorionic Membrane Products”,    -   “Methods of Manufacture of Immunocompatible Chorionic Membrane”        Products,    -   “Immunocompatible Amniotic Membrane Products”,    -   “Methods of Manufacture of Immunocompatible Amniotic Membrane        Products”, and    -   “Therapeutic Products Comprising Vitalized Placental        Dispersions”.

TECHNICAL FIELD

The present invention relates to placental products, methods of medicaltreatment using placental products, and methods of making placentalproducts.

BACKGROUND

The structural integrity of tissue is achieved, in part, by a dynamicinteraction of the tissue with bioactive molecules, extracellularmatrix, and a host of circulating cell types. Such interactions are alsopivotal during tissue aging, injury, restorative and regenerativetreatments. For example, burns produce local tissue damage as well assystemic consequences. Currently, treatment of burn wounds is focused onpromoting healing and decreasing the risk of infection. Burn woundscontinue to be a frustrating and serious problem in the clinic, andthese wounds are often accompanied by high morbidity and mortalityrates. The standard of care for burns includes the use of antisepticsand gauze wound dressings. However, for severe and large surface areaburns, this treatment is not satisfactory. The gold standard for severeburn treatment continues to be autologous living skin grafts. However,the amount of skin available for grafting is often extremely limited,and this procedure always results in donor site wounds.

Attempts to improve burn wound care have included the use of a singlegrowth factor or cocktail of growth factors as well as biological skinsubstitutes. Growth factors such as epidermal growth factor (EGF),platelet derived growth factor (PDGF), basic fibroblast growth factor(FGF), vascular endothelial growth factor (VEGF), and other singularfactors have been tested in burn wound healing; however, with varyingresults.

The use of placental membranes for burns and other types of woundsoriginated more than 100 years ago (reviewed by Kesting et al., 2008).Placental membranes contain components that are present in skin andrequired for wound healing such as extracellular matrix, growth factors,and cells, including MSCs that are responsible for orchestrating theprocess of wound healing. The effectiveness of placental membranes suchas amniotic membranes for burns was recorded in a number of publishedreports; however, the use of placental membranes for large surface areaburns is limited due to challenges in providing sufficient placentalmembranes to cover large areas.

What is needed in the art is a therapeutic product that provides thebenefits of placental membranes yet can be applied in fluid form.Moreover, needed is a product that provides dynamic therapy throughoutmore than one, optimally all, of the phases of wound repair:inflammatory, proliferative, and remodeling.

SUMMARY OF THE INVENTION

The present invention provides methods of manufacturing placentalproducts comprising placental cells and a placental dispersioncomprising placental factors. The placental cells and the placentaldispersion are derived from placental tissue, e.g. a whole placenta orportion thereof. Placental tissue can be obtained by mechanicalmanipulation (e.g. dissection) or enzymatic digestion or combinationsthereof. A placental tissue can optionally be an amnion, chorion, amixture of amnion and chorion, or other tissue described here.

The present invention also provides a method of treating a tissue injury(e.g. wound or burn) comprising administering to a patient in needthereof a placental product of the present invention.

Optionally, the placental dispersion is a homogenate.

Optionally, placental factors present include extracellular matrixcomponents.

Optionally, the placental dispersion comprises one or more placentalfactors set forth in Table 1, Table 2, Table 3, or Table 5.

Optionally, the placental cells comprise stromal cells such as MSCs(mesenchymal stem cells) and PSCs (placental stem cells).

In one embodiment, the method of making a placental product is aparallel processing method that comprises:

-   -   i) obtaining a first placental (e.g. amniotic or chorionic)        tissue;    -   ii) obtaining placental cells from the first placental tissue;    -   iii) obtaining a second placental (e.g. amniotic or chorionic)        tissue;    -   iv) disrupting the second placental tissue to form a dispersion        comprising placental factors;    -   v) combining the placental cells and the dispersion to form the        placental product.

Optionally, the first placental tissue and the second placental tissueare autologous to each other, for example, derived from the same donor.

In one embodiment, the method of making a placental product is a serialprocessing method wherein the second placental tissue is derived fromthe first placental tissue after said step of isolating the placentalcells from a first placental tissue. For example, a first chorionictissue may be retained after isolating a population of cells thereof,and then disrupted to form a dispersion. The dispersion may then becombined with the placental cells.

Optionally, the step of isolating the placental cells comprisescontacting the first placental tissue (e.g. amnion or a chorion or achorion lacking trophoblasts) with a digestive enzyme, such as acollagenase II. Optionally, the first placental tissue is exposed to alimited digestion with an enzyme such as collagenase II; e.g. exposurefor less than about 1 hour (e.g. about 10 minutes or about 20 minutes).

Optionally, the placental tissue (from which the placental dispersion isproduced) is chorionic tissue depleted of trophoblasts by treatment witha digestive enzyme such as dispase II followed by physical removal.

In another embodiment, the method of making a placental productcomprises:

-   -   i) obtaining a placental (e.g. amniotic or chorionic) tissue;    -   ii) exposing the placental tissue to collagenase;    -   iii) dividing the placental tissue into a first portion and a        second portion;    -   iv) isolating placental cells from the first placental portion;    -   v) disrupting the second placental portion to form a dispersion        comprising placental factors; and    -   vi) combining the placental cells and the placental dispersion        to form the placental product.    -   vii) In another embodiment, the method of making a placental        product comprises:    -   viii) obtaining a placental (e.g. amniotic or chorionic) tissue;    -   ix) exposing the placental tissue to a collagenase for a time        sufficient to release placental cells;    -   x) isolating the released placental cells from the collagenase        exposed placental tissue;    -   xi) disrupting the collagenase exposed placental tissue to form        a dispersion comprising placental factors; and    -   xii) combining the placental cells and the placental dispersion        to form the placental product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts cell viability, before and after a freeze-thaw cycle of aplacental product comprising isolated cells and a placental dispersion.

FIG. 2 depicts recovery of viable cells isolated by digestion.

FIG. 3 depicts cell viability, before and after a freeze-thaw cycle of aplacental product comprising isolated cells and a placental dispersion.

FIG. 4 depicts recovery of viable cells isolated by digestion.

FIG. 5 depicts the level of viable cells in a placental product madewith or without a step of cell isolation before tissue disruption.

FIG. 6 depicts cell phenotype of cells in a placental product.

FIG. 7 depicts cell viability using various cryoprotectants

FIG. 8 depicts placental tissue weight and live cells recoveredfollowing collagenase treatment of various incubation times.

FIG. 9 depicts the number of collagenase-released cells from multipledonors.

FIG. 10 depicts viable cell level in a placental product when a placentais subjected to hypoxic or normoxic conditions.

FIG. 11 depicts cell viability when a placenta is subjected to hypoxicor normoxic conditions.

FIG. 12 depicts expression of bFGF in placental products for 14 days inculture.

FIG. 13 depicts expression of IFN-2α and TGF-β3 in placental products,

FIG. 14 BMP-2, BMP-4, BMP-7, PLAB, PIGF, and IGF-1 were detected inplacental products derived from the chorionic membrane

FIG. 15. Representative image of passage 2 cells isolated and expandedfrom a placental product derived from the chorionic membrane

FIG. 16 depicts recovery of viable cells isolated by digestion usingvarious collagenase II enzymes.

FIG. 17 depicts cell viability, before and after a freeze-thaw cycle ofa placental product comprising isolated cells and a placentaldispersion.

DETAILED DESCRIPTION OF THE INVENTION

As used here, the following definitions and abbreviations apply.

-   -   “Chorionic tissue” or “Chorionic membrane” means the chorion or        a portion thereof, e.g. the trophoblast, the somatic mesoderm,        or combinations thereof.    -   “Examplary” (or “e.g.” or “by example”) means a non-limiting        example.    -   “Placental dispersion” means a product formed by        physical/mechanical disruption of placental tissue. For example,        a dispersion may be in the form of a homogenate, a blend, a        suspension, a colloid, or a solution.    -   “Placental tissue” means tissue derived from the placenta in the        broadest sense of the word. Placental tissue can be a whole        placenta or any portion thereof. “Portions of the placenta” is        meant to include chorion, amnion, a chorion and amniotic        membrane (e.g. amnio-chorion), Wharton's jelly, umbilical cord,        placental cotyledons or combinations thereof. The placental        tissue may be dissected or digested (or combinations thereof) to        remove portions, membrane, or structures.    -   “Placental cells” means any cell that can be obtained from a        placenta, without regard to genetic origin (e.g. maternal vs.        fetal), developmental origin (e.g. endodermal, ectodermal, or        mesodermal), or differentiation. Placental cells may comprise        any placental cells known in the art, for example, mesenchymal        stem cells (MSCs), endometrial stromal cells (ESCs),        placenta-derived mesenchymal progenitor cells, placental        mesenchymal stem cells, fibroblasts, epithelial cells, placental        mesenchymal cells, macrophages, and the like.    -   “Placental cells” are further meant to require some feature of        live cells such as one or more of metabolic activity, structural        integrity (e.g. exclusion of a viability stain such as methylene        blue), mitotic activity, signal transduction, and the like.    -   “Placental factor” means any product that is obtainable from a        placental tissue (or placental cells). The product can be an        angiogenic factor, chemokine, cytokine, growth factor, protease,        protease inhibitor, or matrix component. Examplary placental        factors are listed in Table 1, Table 2, Table 3, and Table 5.    -   “Tissue injury” means an injury of any tissue such as skin or        the outer layer of any organ. By injury, it is meant a pathology        that involves or results from an mechanical, metabolic or other        insult. Examples of such tissue injuries are burns, wounds,        ulcerations, and lacerations, ablations (including laser,        freezing, cryo-surgery, heat and electrical ablations), and        surgical incisions.

Placental Product

Overview

It has been surprisingly discovered that a placental product can now beproduced by combining placental cells and a placental dispersion toproduce a medicinal product of substantial and superior therapeuticvalue when administered to a tissue injury. The placental product hasseveral advantageous properties.

Fluidity. The placental product shares certain properties of a fluidsuch as an ability to deform under an applied stress and can bequantified measurements of viscosity. Thus, the present placentalproduct can be spread over the surface of the surface to which it isapplied. For example, one ml of placental product can be spreadtopically to cover more than about any of about 1 cm², about 10 cm²,about 25 cm², about 50 cm², or about 100 cm² of skin. This fluidproperty solves the problem of limited applicability of products thatretain the non-elastic properties of tissue (e.g. skin grafts).Moreover, the fluidity of the present placental product now makes itpractical for new uses such as application to articulating joints andcurved surfaces. It also provides a means of rapid application.

Extended release. Extended release formulations, especially for topicalpharmaceutical products, are especially problematic. Moreover, due tonatural instabilities as well as metabolic degradation, topicalformulations often exhibit substantial loss of activity with time afteradministration. Without being bound by theory, the inventors believethat the placental cells of the present placental products produceplacental components after administration. Thus, the present placentalproducts can contain placental components derived from the placentaldispersion and derived from the placental cells and depletion ofplacental components can be reduced. Additionally, placental cells inthe present placental product can produce placental factors (e.g.protease inhibitors) that reduce the metabolic degradation of placentalfactors.

Dynamic responsivity. Without being bound by theory, the inventorsbelieve that presence of live placental cells provide to the placentalproduct the capacity to respond to physiologic stimuli in a mannersomewhat analogous to endogenous cells in situ. Evidence of dynamicresponsivity includes stimulated release of placental factors or changesin the placental factor profile with time after administration.

Placental Cells

Placental cells may be obtained from any placental tissue (e.g.chorion). Placental cells may be obtained by processing placental tissuein any manner which retains cell viability of at least one cell type(e.g. MSCs). For example, placental cells may be isolated or purifiedfrom placental tissue (e.g. by collagenase digestion of the chorion) ormay be obtained without isolation from one or more placental factors(e.g. extracellular matrix) or from other placental cells.

Placental cells may be obtained by any method known in the art. Usefulmethods of obtaining placental cells (e.g. chorionic cells) aredescribed, for example, by Portmann-Lanz et al. (“Placental mesenchymalstem cells as potential autologous graft for pre- and perinatalneuroregeneration”; American Journal of Obstetrics and Gynecology (2006)194, 664-73), (“Isolation and characterization of mesenchymal cells fromhuman fetal membranes”; Journal Of Tissue Engineering And RegenerativeMedicine 2007; 1: 296-305.), and (Concise Review: Isolation andCharacterization of Cells from Human Term Placenta: Outcome of the FirstInternational Workshop on Placenta Derived Stem Cells”).

In one embodiment, placental cells are obtained by contacting placentaltissue with one or more digestive enzymes, for example, by immersingplacental tissue (e.g. a chorion, or placental tissue lackingtrophoblasts) in a solution containing the digestive enzyme. Thedigestive enzyme may be any digestive enzyme known in the art. Thedigestive enzyme may also be combination of enzymes. Examplary digestiveenzymes include one or more: collagenases (e.g., collagenase I, II, Illand IV), matrix metalloprotease, neutral proteases, papains,deoxyribonucleases, serine protease (e.g. trypsin, chymotrypsin,elastase), or any combination thereof.

In one embodiment, placental cells are obtained from a chorion bycontacting a chorion (e.g. a chorion lacking trophoblasts) with acollagenase (e.g. collagenase II). The collagenase may present in anysuitable concentration, for example, about 100 U/mL to about 1000 mL,and in any suitable collagenase solvent, such as DMEM, and at anysuitable temperature, for example 37° C. The chorion may be contactedwith the digestive enzyme for any suitable period of time. Optionally,the chorion is contacted with a collagenase (e.g. collagenase II) forless than about any of: about 3 hrs, about 2 hr, or about 1 hr.Optionally, the chorion is contacted with the collagenase (e.g.collagenase II) for less than about 1 hour, for example, less than aboutany of: about 60 min, about 50 min, about 40 min, about 30 min, about 20min, about 15 min, about 10 min, or about 5 min. Optionally, the chorionis contacted with a collagenase for a limited period of time such that asubstantial portion of the placental tissue is retained on a about 100micron filter. Optionally, the chorion is contacted with collagenase IIfor a limited period of time such that a substantial portion of theplacental tissue is retained on a 100 micron filter. Optionally, afterthe placental cells are obtained, the chorion is disrupted to form adispersion and the population is combined with (e.g. added to) thedispersion.

Surprisingly, a step of obtaining placental cells before subjecting theplacental tissue to tissue disruption results in substantially a greaternumber of cells generally and also results in a population of cells thatmore resemble the population in the placental tissue than population ofcells that are obtained from disrupted placental tissue.

A placental product that comprises placental cells from placental tissuethat has not been disrupted surprisingly provides a therapeuticallyeffective amount of viable cells without the need for ex vivo expansionof the placental cells. Although ex vivo expansion is a known method ofincreasing the number of viable cells in a population, such a step oftenleads to changes in the population make-up or distribution of cellphenotype. For example, various cells in a population may expand atdifferent rates and expansion may also induce differentiation.Accordingly, one embodiment of the present invention provides aplacental product comprising placental cells derived from a placentaltissue wherein the placental cells exhibits a phenotypic distribution ofcells which is substantially similar to the cells of the placentaltissue of origin.

Placental Dispersion

A placental dispersion may be provided by disrupting a placenta (e.g. achorion). The disruption of placental tissue may be accomplished by anyphysical/mechanical method of disrupting tissue (i.e. use of a “tissuedisruptor” or “means for disruption”). For example, disruption maycomprise homogenization, maceration, use of a blender, crushing, ormincing. Disruption may additionally or alternatively comprise shearing,mincing, dicing, or chopping. Disruption may additionally oralternatively comprise sonication.

The placental tissue may be disrupted for any suitable duration whichproduces a dispersion from the placenta. For example, the placenta maybe disrupted (e.g. homogenized) for less than about 20 sec, about 15sec, about 10 sec, or about 5 seconds.

The placental tissue can be disrupted sufficient to form a placentalproduct with fluid characteristic and yet retain viable cells.Accordingly, live cells in the placental products of the presentinvention can additionally comprise placental cells that are derivedfrom the placental dispersion.

The extent of tissue disruption may be reduced by a prior enzymaticdigestion step with a matrix degrading enzyme such a collagenase(s), aprotease(s), or combinations thereof. Indeed, it has surprisingly beendiscovered that such prior digestion preserves viable cells in theplacental dispersion. For example, the length of treatment by a tissuedisruptor can be reduced by prior enzymatic digestion.

Placental Factors

A placental product of the present invention may comprise one or moreplacental factors where the placental factors are components of theplacental dispersion or components released into the placental productby the placental cells or a combination thereof.

It has surprisingly been discovered that the content of placentalfactors in placental products made according to the present inventionhave an unexpected therapeutic value. Such content of placental factorsas taught herein is accordingly referred to here as a “therapeuticprofile”.

In one embodiment of the present invention, a therapeutic profile is onethat provides two or more, or three or more, or four or more placentalfactors listed in Table 1, Table 2, Table 3, or Table 5. Optionally, theplacental factors are present in an amount of about 20% to about 500% ofthe mean concentration set forth in Table 1, Table 2, or Table 5.Optionally, the placental factors are present in an amount of about 20%to about 500% of the minimum and the maximum (respectively) of thevalues set forth in Table 1, Table 2, or Table 5

Placental factors, according to the present invention, can beplacental-derived factors such as angiogenic factors, chemokines,cytokines, growth factors, matrix metalloproteases, extracellular matrixproteins (or “matrix proteins”), and combinations thereof. The presentplacental products can comprise any of these placental factors.

The present placental products can optionally comprise a therapeuticprofile of one or more of a PDGF (e.g. PDGF-bb), EGF, FGF, TGF-β1,TGF-β3, and VEGF and/or one or more of IL-8, IL-6, and MCP-1.

Useful placental products of the present invention can have atherapeutic profile as set forth in Table 1, Table 2, Table 3, or Table5.

Useful placental products of the present invention can have atherapeutic profile comprising at least 25% of the minimum concentrationof one or more placental factors set forth in Table 1 and optionally nomore than 400% of the maximum concentration of one or more placentalfactors set forth in Table 1. In one embodiment, the one or moreplacental factors comprise fibronectin, TIMP, TGFβ1, bFGF, and MMPs(e.g. MMP1, 2, 4, 7, 8, 9, and 10).

Useful placental products of the present invention can have atherapeutic profile comprising four or more placental factors where atleast two placental factors are extracellular matrix components (orfragment thereof).

Placental products of the present invention can comprise a therapeuticprofile of one or more placental factors which promote the migration ofepithelial cells into a wound area (e.g. HGF and/or KGF), optionally incombination with a growth factor such as TGF-β1. Optionally theconcentration of such placental factors is about 25% of the minimumvalues set forth in Table 1 and optionally no more than 400% of themaximum concentration set forth in Table 1

Placental products can comprise a therapeutic profile of placentalfactors that are mitotic or growth promoting. Placental products cancontain HGF and KGF. For example, HGF at a concentration of about 5,000to about 200,000 pg/mL and KGF at a concentration of about 5,000 toabout 400,000 pg/mL are present in an examplary placental product asdetailed in Example 10. Optionally, such placental products are usefulin preventing scaring or a useful therapy aid duringre-epithelialization,

Placental products of the present invention can comprise a therapeuticprofile of placental factors comprising one or more angiogenic factors(e.g. VEGF and/or bFGF) and can optionally additionally comprise one ormore growth factors (e.g. TGF-β1 and/or TGF-β2),

Examplary placental products of the present invention contain atherapeutic profile of VEGF levels greater than about 10 pg/ml orgreater than about 50 pg/ml or greater than about 100 pg/ml. Forexample, an examplary placental product can comprise greater than about200 pg/ml as detailed in Example 10.

Examplary placental products of the present invention contain atherapeutic profile of bFGF levels greater than any of about 10 or 100or 1,000 or 10,000 pg/ml. An examplary placental product can comprisegreater than about 11,000 pg/mL, as detailed in Example 10. Optionallysuch FGF-comprising placental products are useful for burn woundhealing.

Placental products of the present invention can comprise a therapeuticprofile of TGF-β1 and TGF-β2. An examplary placental product, asdetailed in Example 10, comprises bFGF, TGF-β1 and TGF-β2. Optionally,such placental products are useful when the skin pathology being treatedinvolves an inflammatory or a scaring pathology.

Placental products of the present invention may comprise a therapeuticprofile of one or more protease inhibitors, such as tissue inhibitors ofmatrix metalloproteinases (TIMPs), alpha-2 macroglobulin, and/orthrombospondins.

In one embodiment, a placental product (e.g. derived from chorion)comprises one or more protease inhibitors.

In one embodiment, a placental product (e.g. derived from chorion)comprises one or more protease inhibitors and extracellular matrixproteins

In one embodiment, a placental product (e.g. derived from chorion)comprises one or more protease inhibitors and viable cells.

In one embodiment, a placental product (e.g. derived from chorion)comprises one or more protease inhibitors, extracellular matrixproteins, and viable cells.

Without being bound by theory, the present inventors believe that thesurprising efficacy that characterizes placental products of the presentinvention result in an interaction between the placental cells and theplacental factors comprising (1) growth factor(s), (2) proteaseinhibitor(s), and (3) extracellular matrix components. Growth factorscan bind to extracellular matrix thereby protecting the growth factorsfrom degradation and effectively extending the half life of the growthfactors. Bioavailability can be further regulated by subsequent releaseor matrix degradation. Similarly, protease inhibitors in examplaryplacental products provide additional protection against proteasedegradation. The placental cells further can protect growth factors andother placental factors in the placental products from degradation byproviding additional protease inhibitors and growth factors.Accordingly, such placental products can optionally maintain surprisingproduct integrity for extended periods of time resulting in placentalproducts that require less frequent applications and superior treatmentof tissue injuries such as burns and wounds. Surprisingly, the growthfactors in such placental products can demonstrate a longer half-life incomparison to other growth factor therapies such as ACCS.

Formulation

The placental products of the present invention are administered as adermatologically acceptable pharmaceutical product. Optionally, activepharmaceutical ingredients or excipients or combinations thereof can beadded.

Viscosity. Viscosity values that are useful and desirable according tothe present invention vary as a function of the indication beingtreated. For example, where broad coverage (i.e. large areas of skin) orlower concentrations of placental products are desired, a less viscousformulation is advantageous. Examples of less viscous formulations arethose of about 1,000 cps to about 50,000 cps, or about 2,000 cps toabout 25,000 cps, or about 2,000 cps to about 10,000 cps, or about 5,000cps to about 15,000 cps. Such less viscous compositions facilitatespreading of applied composition.

Where more restricted coverage or higher levels of placental productsare desired, a more viscous formulation is advantageous. Examples ofmore viscous formulations are about 20,000 cps to about 200,000 cps orabout 50,000 cps to about 100,000 cps.

The skilled artisan will now readily recognize that the desiredviscosity can be attained according to the present invention byadjustments of the dispersion method (discussed elsewhere herein) or byselection of a dermatologically acceptable thickening agent andempirically determining the concentration necessary to achieve thedesired thickening agent.

The placental products of the present invention can optionally includeone or more antibiotic, emollient, keratolytics agent, humectants,anti-oxidants, preservative, or combinations thereof.

In one embodiment, a placental product comprises albumin, such as HSA orBSA. Optionally, the placental product comprises an electrolytesolution, for example, to provide physiological osmolality and pH (e.g.Plasma-LyteA). Optionally, the placental product comprises acryopreservative, such as DMSO, glycerol, sericin, sugars, or a mixturethereof.

In one embodiment, a placental product comprises albumin, an electrolytesolution, and a cryopreservative. Optionally, the therapeutic productcomprises 1% to about 15% albumin by weight and about 5% to about 20%cryopreservative by volume (e.g. about 10%). Optionally, the albumin isHSA, the electrolyte solution is Plasma-Lyte A, and the cryopreservativeis DMSO.

Manufacture

Overview

A placental product of the present invention may be manufactured from aplacenta in any suitable manner that provides the technical featurestaught herein. Any placental tissue is useful according to the presentinvention. Each of the embodiments of the present invention set forthhere are meant to specifically embrace placental products where theplacental dispersion is a dispersion of chorion that is depleted of orlacking trophoblastic components.

According to the present invention, the placenta is processed to producethe placental dispersion and the placental cells.

In one embodiment, the placental dispersion and the placental cells arederived from a different placenta or different placental portion (e.g.parallel processing).

In one embodiment, the placental dispersion and the placental cells arederived from the same placenta or the same placental portion (e.g.sequential processing).

Manufacturing Method 1. In one embodiment, a placental product ismanufactured by:

-   -   obtaining a placental (e.g. chorionic) tissue;    -   digesting the placental tissue with one or more matrix degrading        enzymes (e.g. a collagenase, optionally collagenase II);    -   obtaining placental cells from the digested placental tissue;    -   disrupting the digested placental tissue with a tissue disruptor        to form a placental dispersion comprising placental factors; and    -   combining the placental cells and the placental dispersion to        form the placental product.

Optional Manufacturing Method 2 In one embodiment, a placental productis manufactured by:

-   -   obtaining a first placental (e.g. chorionic) tissue;    -   digesting the first placental tissue with one or more matrix        degrading enzymes (e.g. a collagenase, optionally collagenase        II);    -   obtaining placental cells from the digested first placental        tissue;    -   obtaining a second placental tissue;    -   disrupting the second placental tissue with a tissue disruptor        to form a placental dispersion comprising placental factors; and    -   combining the placental cells and the placental dispersion to        form the placental product.

For either Manufacture Method, the placental tissue can be a choriontissue such as a chorion tissue that has been processed to reduce thenumber of trophoblastic cells.

Examplary placental products of the present invention can bemanufactured or provided with a bandage or wound dressing.

Trophoblast Removal

In one embodiment, trophoblasts are depleted or removed to produce theplacental tissue from which the placental cells or the placentaldispersion or both are derived. Surprisingly, such a placental producthas one or more of the following superior features:

-   -   a. is substantially non-immunogenic;    -   b. provides remarkable healing time; and    -   c. provides enhanced therapeutic efficacy.

Trophoblasts may be removed in any suitable manner which substantiallydiminishes the trophoblast content of the placental product. Optionally,the trophoblasts are selectively removed or otherwise removed withouteliminating a substantial portion of one or more therapeutic componentsfrom the placenta (e.g. MSCs, placental factors, etc). Optionally, thetrophoblasts are removed before isolating a population of cells and/ordisrupting the placental tissue.

One method of removing trophoblasts comprises treating the placenta(e.g. chorion or amnio-chorion) with a digestive enzyme such as dispase(e.g. dispase II) and separating the trophoblasts from the placenta.Optionally, the step of separating comprises mechanical separation suchas scraping. Optionally, scraping comprises scraping with a softinstrument such as a finger.

Useful methods of removing trophoblasts from a placenta (e.g. chorion)are described by Portmann-Lanz et al. (“Placental mesenchymal stem cellsas potential autologous graft for pre- and perinatal neuroregeneration”;American Journal of Obstetrics and Gynecology (2006) 194, 664-73),(“Isolation and characterization of mesenchymal cells from human fetalmembranes”; Journal Of Tissue Engineering And Regenerative Medicine2007; 1: 296-305.), and (Concise Review: Isolation and Characterizationof Cells from Human Term Placenta: Outcome of the First InternationalWorkshop on Placenta Derived Stem Cells”).

Preservation

A placental product of the present invention may be used fresh or may bepreserved for a period of time.

Also as depicted in FIG. 1, a placental product of the presentinvention, cell viability is retained surprisingly well after afreeze-thaw cycle

In one embodiment, a placental product is cryopreserved. A placentalproduct may be cryopreserved by freezing (e.g. a −80° C.). Freezing maycomprise storage in a cryopreservation medium such as DMSO, glycerol,sericin, sugars, or mixtures thereof. Freezing may comprise, forexample, incubating the placental product at 4° C. for 30-60 min, andthen incubating at −80° C. until use. The placental product may then bethawed for use.

A placental product may be formulated in a cryopreservative beforecryopreservation. Examplary cryopresevatives include DMSO, glycerol, andthe like. The cryopreservative may further be formulated with additionalcomponents such as albumin (e.g. HSA or BSA), an electrolyte solution(e.g. Plasma-Lyte), or a combination thereof. Optionally, the placentalproduct comprises 1% to about 15% albumin by weight and about 5% toabout 20% cryopreservative by volume (e.g. about 10%).

Optionally, a placental product can be formed by the addition ofcryopreserved placental cells of the present invention to a fresh (neverfrozen) placental dispersion or to a frozen placental dispersion or to alyophilized placental dispersion.

Optionally, a placental product can be formed by the addition of freshplacental cells of the present invention to a frozen placentaldispersion or to a lyophilized placental dispersion.

Methods of Use

The placental products of the present invention may be used to treat anytissue injury. A method of treatment may be provided, for example, byadministering to a subject in need thereof, a placental product of thepresent invention.

A typical administration method of the present invention is topicaladministration. Administering the present invention can optionallyinvolve administration to an internal tissue where access is gained by asurgical procedure.

Placental products can be administered autologously, allogeneically orxenogeneically.

In one embodiment, a present placental product is administered to asubject to treat a wound. Optionally, the wound is a laceration, scrape,thermal or chemical burn, incision, puncture, or wound caused by aprojectile. Optionally, the wound is an epidermal wound, skin wound,chronic wound, acute wound, external wound, internal wounds, congenitalwound, ulcer, or pressure ulcer. Such wounds may be accidental ordeliberate, e.g., wounds caused during or as an adjunct to a surgicalprocedure. Optionally, the wound is closed surgically prior toadministration.

In one embodiment, the injury is a burn. Optionally, the burn is afirst-degree burn, second-degree burn (partial thickness burns), thirddegree burn (full thickness burns), infection of burn wound, infectionof excised and unexcised burn wound, loss of epithelium from apreviously grafted or healed burn, or burn wound impetigo.

In one embodiment, the injury is an ulcer, for example, a diabetic ulcer(e.g. foot ulcer).

In one embodiment, a placental product is administered by placing theplacental product directly over the skin of the subject, e.g., on thestratum corneum, on the site of the wound, so that the wound is covered,for example, using an adhesive tape. Additionally or alternatively, theplacental product may be administered as an implant, e.g., as asubcutaneous implant.

In one embodiment, a placental product is administered to the epidermisto reduce rhytids or other features of aging skin. Such treatment isalso usefully combined with so-called cosmetic surgery (e.g.rhinoplasty, rhytidectomy, etc.).

In one embodiment, a placental product is administered to the epidermisto accelerate healing associated with a dermal ablation procedure or adermal abrasion procedure (e.g. including laser ablation, thermalablation, electric ablation, deep dermal ablation, sub-dermal ablation,fractional ablation, and microdermal abrasion).

Other pathologies that may be treated with placental products of thepresent invention include traumatic wounds (e.g. civilian and militarywounds), surgical scars and wounds, spinal fusions, spinal cord injury,avascular necrosis, reconstructive surgeries, ablations, and ischemia.

In one embodiment, a placental product of the present invention is usedin a tissue graft procedure. Optionally, the placental product isapplied to a portion of the graft which is then attached to a biologicalsubstrate (e.g. to promote healing and/or attachment to the substrate).By way of non-limiting example, tissues such as skin, cartilage,ligament, tendon, periosteum, perichondrium, synovium, fascia, mesenterand sinew can be used as tissue graft.

In one embodiment, a placental product is used in a tendon or ligamentsurgery to promote healing of a tendon or ligament. Optionally, theplacental product is applied to portion of a tendon or ligament which isattached to a bone. The surgery can be any tendon or ligament surgery,including, e.g. knee surgery, shoulder, leg surgery, arm surgery, elbowsurgery, finger surgery, hand surgery, wrist surgery, toe surgery, footsurgery, ankle surgery, and the like. For example, the placental productcan be applied to a tendon or ligament in a grafting or reconstructionprocedure to promote fixation of the tendon or ligament to a bone.

Through the insight of the inventors, it has surprisingly beendiscovered that placental products of the present invention providesuperior treatment (e.g. healing time and/or healing strength) fortendon and ligament surgeries. Tendon and ligament surgeries can involvethe fixation of the tendon or ligament to bone. Without being bound bytheory, the present inventors believe that osteogenic and/orchondrogenic potential of MSCs in the present placental productspromotes healing process and healing strength of tendons or ligaments.The present inventors believe that the present placental productsprovide an alternative or adjunctive treatment to periosteum-basedtherapies. For example, useful periosteum based treatments are describedin Chen et al. (“Enveloping the tendon graft with periosteum to enhancetendon-bone healing in a bone tunnel: A biomechanical and histologicstudy in rabbits”; Arthroscopy. 2003 March; 19(3):290-6), Chen et al.(“Enveloping of periosteum on the hamstring tendon graft in anteriorcruciate ligament reconstruction”; Arthroscopy. 2002 May-June;18(5):27E), Chang et al. (“Rotator cuff repair with periosteum forenhancing tendon-bone healing: a biomechanical and histological study inrabbits”; Knee Surgery, Sports Traumatology, Arthroscopy Volume 17,Number 12, 1447-1453), each of which are incorporated by reference.

As non-limiting example of a method of tendon or ligament surgery, atendon is sutured to and/or wrapped or enveloped in a placental membraneand the tendon is attached to a bone. Optionally, the tendon is placedinto a bone tunnel before attached to the bone.

In one embodiment, the tendon or ligament surgery is a graft procedure,wherein the placental product is applied to the graft. Optionally, thegraft is an allograft, xenograft, or an autologous graft.

In one embodiment, the tendon or ligament surgery is repair of a tornligament or tendon, wherein the placental product is applied to the tornligament or tendon.

Non-limiting examples of tendons to which a placental product can beapplied include a digitorum extensor tendon, a hamstring tendon, a biceptendon, an Achilles Tendon, an extensor tendon, and a rotator cufftendon.

In one embodiment, a placental product of the present invention is usedto reduce fibrosis by applying the placental product to a wound site.

In one embodiment, a placental product of the present invention is usedas an anti-adhesion wound barrier, wherein the placental product isapplied to a wound site, for example, to reduce fibrosis (e.g.postoperative fibrosis).

Non-limiting examples of wound sites to which the placental product canbe applied include those that are surgically induced or associated withsurgery involving the spine, laminectomy, knee, shoulder, or childbirth, trauma related wounds or injuries, cardiovascular procedures,angiogenesis stimulation, brain/neurological procedures, burn and woundcare, and ophthalmic procedures. For example, optionally, the wound siteis associated with surgery of the spine and the stromal side of theplacental product is applied to the dura (e.g. the stromal side facingthe dura). Direction for such procedures, including the selection ofwound sites and/or methodologies, can be found, for example, in WO2009/132186 and US 2010/0098743, which are hereby incorporated byreference.

A placental product of the present invention can optionally be used toreduce adhesion or fibrosis of a wound. Postoperative fibrosis is anatural consequence of all surgical wound healing. By example,postoperative peridural adhesion results in tethering, traction, andcompression of the thecal sac and nerve roots, which cause a recurrenceof hyperesthesia that typically manifests a few months after laminectomysurgery. Repeated surgery for removal of scar tissue is associated withpoor outcome and increased risk of injury because of the difficulty ofidentifying neural structures that are surrounded by scar tissue.Therefore, experimental and clinical studies have primarily focused onpreventing the adhesion of scar tissue to the dura matter and nerveroots. Spinal adhesions have been implicated as a major contributingfactor in failure of spine surgery. Fibrotic scar tissue can causecompression and tethering of nerve roots, which can be associated withrecurrent pain and physical impairment.

The placental products disclosed herein are useful in treating a numberof wounds including: tendon repair, cartilage repair (e.g. femoralcondyle, tibial plateau), ACL replacement at the tunnel/bone interface,dental tissue augmentation, fistulas (e.g. Crohn's disease, G-tube,tracheoesophogeal), missing tissue at adhesion barriers (e.g. nasalseptum repair, vaginal wall repair, abdominal wall repair, tumorresection), dermal wounds (e.g. partial thickness burns, toxic epidermalnecrolysis, epidermolysis bullosa, pyoderma gangrenosum, ulcers e.g.diabetic ulcers (e.g. foot), venous leg ulcers), surgical wounds, herniarepair, tendon repair, bladder repair, periosteum replacement, keloids,organ lacerations, epithelial defects, and repair or replacement of atympanic membrane.

The presently described technology and its advantages will be betterunderstood by reference to the following examples. These examples areprovided to describe specific embodiments of the present technology. Byproviding these specific examples, it is not intended limit the scopeand spirit of the present technology. It will be understood by thoseskilled in the art that the full scope of the presently describedtechnology encompasses the subject matter defined by the claimsappending this specification, and any alterations, modifications, orequivalents of those claims.

The citations provided herein are hereby incorporated by reference forthe cited subject matter.

In the present specification, use of the singular includes the pluralexcept where specifically indicated.

EXAMPLES Example 1 Obtaining Placental Tissue

A whole placenta was obtained from a registered tissue bank afterinformed consent. The placenta and placed, with the maternal surface(rough surface) face down, on a sterile tray. The amniotic-chorionicmembrane was cut and removed from the placenta. The chorionic membranewas then separated from the amnion and washed twice in PBS.

The chorionic membrane was then soaked in an anticoagulant (ACD-A)solution to remove blood clots and then washed again in PBS.

The chorionic membrane was then digested by incubation with dispase IIfor 30 min. at 37° C. The trophoblast layer was mechanically removed byscraping with fingers and the chorion was washed again in PBS.

The chorionic membrane was then incubated for 24 hours in an antibioticcocktail containing gentamicin, vancomycin, and amphotericin B, andwashed again in PBS.

Example 2 Digesting Placental Tissue

A chorion membrane (obtained from Example 1) was digested by incubationin 200 mL of a collagenase II solution (300 U/mL in DMEM) for 10 min at37° C. The chorionic membrane was then removed, leaving a digestionsuspension containing collagenase and placental cells.

The volume and container for digestion was determined based on the needto provide a suitable digestion environment for the tissue once placedon a shaker. The digestion was carried out on a standard plate shakerset at moderate speed in a 37° C. cell culture incubator.

Example 3 Obtaining Placental Cells

A digestion suspension comprising placental cells (obtained from Example2) was centrifuged at 2000 rcf for 5 min to separate the digestiveenzyme (collagenase II) from the placental cells. This stepcentrifugation step may enhance cell viability by preventingover-digestion and ensure that the enzyme is washed away beforehomogenizing the tissue. This centrifugation step pellets the cellswithout damaging them, allowing the collagenase II to be removed assupernatant.

The cells were then centrifuged again, the supernatant poured off, andthe placental cells were resuspended in a small volume (2 mL) ofcryprotectant (5% DMSO in saline). Two mL provides an adequate volume toresuspend the cells while not over-diluting the chorion membranedispersion once the cells have been added.

Example 4 Obtaining a Placental Dispersion

A chorionic membrane (obtained from Example 2) was washed twice in PBSto remove residual digestion enzyme and placed in a homogenizationcontainer with 1 ml cryoprotectant per gram of chorionic membrane. Thisvolume was determined to be appropriate for diluting the chorionmembrane enough to produce a dispersion of ideal consistency whilemaintaining protein concentration at clinically significant levels.

The temperature of the chorionic membrane was reduced by placing thecontainer on ice for greater than 10 min. The chorionic membrane wasthen homogenized twice at high speed for 5 sec. using a tissuehomogenizer to obtain a chorionic dispersion (homogenate).

Once the chorion membrane is subjected to digestion, it becomes easy tohomogenize. Surprisingly, only a small amount of homogenization isneeded to create a homogenous solution ideal for clinical use andincreases the amount of live cells present in the final dispersion.

Example 5 Providing a Placental Product

A placental dispersion (obtained from Example 4) was combined withviable isolated placental cells (obtained from Example 3) and mixedthoroughly to provide a placental product. The placental product may beused (e.g. for therapy) fresh or may first be preserved (e.g.cryogenically) for a period of time.

Example 6 Cryopreservation

A placental product (obtained from Example 5) was aliquoted into vialsand incubated at 4° C. for 30-60 min. The vials were then frozen at −80°C. until use.

Example 7 Isolation of Cells without Complete Digestion of Placenta

The inventors tested whether a limited collagenase II digestion might beperformed to obtain a suspension containing live cells and yet preservethe integrity of the placental tissue (e.g. preserve placental factorsand remaining live cells). A brief 10 minute digestion with collagenaseII left the tissue intact and made further handling possible. Inaddition, a 10 min. collagenase digestion was able to produce highnumbers of viable cells

Two placentas were obtained, each from a different donor, and processedaccording to the procedure detailed in Example 1 through Example 2,except a collagenase II concentration of 244 U/mL, as described above. Acell count was performed immediately following digestion to determinethe number of viable cells per gram of tissue that each enzyme was ableto digest away off the tissue. The data are presented in FIG. 2.

The placentas were further processed as described in Example 3 throughExample 6. Before freezing and after thawing, cells were counted using ahemocytometer and trypan blue staining was used to distinguish livecells. The data are presented in FIG. 3.

Surprisingly, a substantial population of cells was isolated bydigestion of less than 1 hr (e.g. 10 min). Digesting the tissue for only10 min allowed the loosening and removal cells from the tissue withoutcompletely breaking up the tissue. In this manner, it was possible toseparate the collagenase II/cell mixture from the chorionic membrane.The inventors discovered that 10 min was an adequate amount of digestiontime and allowed for variances introduced as a result of donorvariability. The digestion process allows isolation of as many livecells as possible while not disrupting the tissue integrity of thechorion membrane to a degree that makes it impossible to manipulatefurther. The chorion membrane could then be disrupted to produce aplacental dispersion that was rich in placental factors while the cellscould be isolated from the enzyme solution and then reintroduced to thedispersion to form the placental product.

Example 8 Isolation of Cells without Complete Digestion of PlacentalTissue

Multiple placental products were prepared and cell counts were takenimmediately following digestion (FIG. 4) and before freezing and afterthawing (FIG. 1), using the procedure described in Example 7. Cells werecounted using a hemocytometer and trypan blue staining was used todistinguish live cells. All cell count data was pooled and a mean wascalculated.

As depicted in FIG. 4, digestion of an intact membrane as taught hereinproduces a surprising number of cells, and does so without mechanicaldisruption of the membrane. Also depicted in FIG. 4, digestion of amembrane as taught herein produces a surprisingly high ratio of viableto non-viable cells.

As depicted in FIG. 1, a fresh placental product of the presentinvention comprises surprisingly high cell viability. Also as depictedin FIG. 1, a placental product of the present invention subjected to afreeze-thaw cycle comprises surprisingly high cell viability. Also asdepicted in FIG. 1, a placental product of the present invention, cellviability is retained surprisingly well after a freeze-thaw cycle.

Example 9 Isolation of Placental Cells

Manufacturing methods were explored to obtain superior recovery of livecells in the placental dispersion. Specifically, an experiment wasperformed to determine the level of viable cells in a placental productmanufactured with or without a step of cell isolation beforehomogenization. Briefly, a placenta prepared according to the proceduredetailed in Example 1. The resulting chorion membrane was then dividedinto equal halves. Half the tissue was processed as described in Example2 through Example 5 and the other half was processed in the same mannerbut without cell isolation (collagenase II digestion) prior tohomogenization followed by recombining the isolated cells with thedispersion. Cells were counted using a hemocytometer and trypan bluestaining was used to distinguish live cells. The data are presented inFIG. 5.

Results indicate that without prior digestion, homogenization eliminatesvirtually all viable cells from the end dispersion. Surprisingly, aplacental product contains a substantially greater number of viablecells and is provides enhanced therapeutic efficacy when manufacturedwith a step of cell isolation before homogenization.

Example 10 Profile of a Placental Product

Multiple placental products were prepared, each from a different donor,according to the procedure detailed in Example 1 through Example 6 andplacental factors were analyzed. Briefly, 1 mL of homogenate from eachplacental product was centrifuged at 14,000 rpm in a microcentrifuge for10 min.

The resulting supernatant from each sample was collected as a testsample. Negative control samples consisted of 5% DMSO in saline(cryopreservation solution) and positive control samples consisted ofcryopreservation solution with a known concentration of spikedrecombinant proteins (bFGF, EGF, and VEGF). Protein profiles comprisingplacental factors listed in Table 1 were obtained using the SearchLightprotein array assay (Aushon Biosystems). Results are indicated in Table1 as a minimum and maximum expression levels (pg/mL) in a pool of fourdonors. Since the supernatant is analyzed rather than the completehomogenate, it is likely that protein level estimates are below actualconcentrations in each chorion membrane homogenate containing livecells. The levels of VEGF and bFGF in each sample were confirmed byELISA.

Surprisingly, many placental factors were detectable at levels that areknown to be influential for burn wound healing as well as in thetreatment of other indications.

As seen from the data in Table 1, placental products of the presentinvention comprise a therapeutic profile of placental factors.

Table 2 sets forth a therapeutic profile of placental products. Onlynow, with the teaching herein, the skilled artisan can examine theplacental factors, consider the functional role as set forth in Table 3,and assess the value of a placental factor in wound repair.

TABLE 1 Therapeutic Profile of Factors in the Placental Products Min.Max. Mean Protein (pg/mL) (pg/mL) (pg/mL) Function MMP1 2210.07 3468.942808.12 Matrix and growth factor degradation, facilitate cell migrationMMP2 8207.46 70964.65 25648.74 MMP3 241.76 615.23 454.49 MMP7 79.784429.02 1190.31 MMP8 778.03 4661.35 2821.20 MMP9 32879.10 149579.1071487.03 MMP10 6728.94 22686.00 14688.40 MMP13 TLTD TLTD TLTD TIMP118739.41 315870.30 116341.69 Inhibit activity of MMPs, angiogenic TIMP27160.87 60711.15 21335.46 TSP1 TLTD TLTD TLTD Regulate TGFβ activity,anti-angiogenic TSP2 1123.02 18784.67 6190.03 TGFα TLTD TLTD TLTDStimulate growth and migration TGFβ1 1041.50 6572.83 2661.65 Promoteangiogenesis, also proliferative and migration stimulatory effects TGFβ291.81 1809.81 558.53 Promote angiogenesis, also proliferative andmigration stimulatory effects TGFβ3 77.02 146.31 104.35 Inhibit scarformation bFGF (FGF-2) 3554.58 11856.91 7479.40 Promote angiogenesis,also proliferative and migration stimulatory effects KGF (FGF-7) 14.15111.58 45.72 Stimulate cell growth and migration EGF 0.42 3.72 1.57Stimulate cell growth and migration HB-EGF TLTD TLTD TLTD PDGFAA 39.20173.52 77.46 Promote angiogenesis, also proliferative and migrationstimulatory effects PDGFAB 495.90 495.90 495.90 PDGFBB 7.73 235.85 70.56VEGF 13.95 211.17 76.73 Promote angiogenesis, also proliferative andmigration stimulatory effects VEGFC 64.77 178.51 118.71 VEGFD 64.7385.55 77.34 HGF 9180.77 71280.10 27480.10 Inhibit scar formation,stimulate cell growth and migration PEDF 805.18 805.18 805.18 Stimulategrowth and migration ANG2 TLTD TLTD TLTD Stimulate growth and migrationIGFBP1 5022.96 1227128.50 322596.69 Regulate IGF and its proliferativeeffects IGFBP2 564.62 564.62 564.62 IGFBP3 226.20 809.16 603.93 ACRP306403.34 33898.70 16229.15 Regulate growth and activity of keratinocytesFibronectin 2950999.50 90198200.00 24973399.00 ECM, cellular adhesion,stimulates growth and migration Alpha2mac 280783.30 4653881.001554151.49 Inhibit protease activity, coordinate growth factorbioavailability IL1ra 961.93 10035.52 3568.27 Anti-inflammatory NGAL420.82 2908.38 1592.17 Anti-bacterial SDF1b TLTD TLTD TLTD Recruit cellsfrom circulation to site of tissue damage TLTD = too low to detect

TABLE 2 Therapeutic Profile of Factors in the Chorionic Membrane Max.Mean Protein Min. (pg/mL) (pg/mL) (pg/mL) MMP1 2882.87 6582.26 4732.56MMP2 748.82 949.52 849.17 MMP3 TLTD TLTD TLTD MMP7 4.46 9.07 6.76 MMP8TLTD TLTD TLTD MMP9 1259.30 2676.23 1967.77 MMP10 79.31 87.51 83.41MMP13 TLTD TLTD TLTD TIMP1 17419.86 50712.30 34066.08 TIMP2 640.73779.98 710.36 TGFα TLTD TLTD TLTD bFGF (FGF-2) 351.28 375.05 363.17 KGF(FGF-7) 1.53 3.07 2.30 EGF 0.75 0.75 0.75 HB-EGF 15.40 84.49 49.94PDGFAA 35.25 39.79 37.52 PDGFAB 14.03 14.43 14.23 PDGFBB 1.29 3.99 2.64VEGF 8.39 125.16 66.78 VEGFC 51.74 123.45 87.60 VEGFD 14.99 20.42 17.70HGF 29979.57 50392.75 40186.16 PEDF TLTD TLTD TLTD ANG2 TLTD TLTD TLTDIGFBP1 934.03 1443.63 1188.83 IGFBP2 134.61 135.86 135.24 IGFBP3 4571.5111970.15 8270.83 LIF TLTD TLTD TLTD GCSF 0.74 1.22 0.98 TPO TLTD TLTDTLTD PIGF TLTD TLTD TLTD ACRP30 225.35 1213.70 719.52 Alpha2mac 8174.449968.59 9071.52 IL1ra 525.53 5168.21 2846.87 NGAL 229.72 938.51 584.11SDF1b TLTD TLTD TLTD TLTD = too low to detect

TABLE 3 Functions of Placental Factors Specific Proteins SelectedFunctions Matrix Metalloproteinase 1 (MMP1), MMP2, 3, 7, 8, 9, 10, 13Matrix and growth factor degradation, facilitate cell migration TissueInhibitors of MMPs (TIMP1 and TIMP2) Inhibit activity of MMPs,angiogenic Angiotensin-2 (Ang-2), Heparin-Bound Epidermal GrowthStimulate growth and migration Factor (HB-EGF), EGF, FGF-7 (also knownas Keratinocyte Growth Factor-KGF), Placenta Growth Factor (PIGF),Pigment Epithelium Derived Factor (PEDF), Thrombopoietin (TPO),Transforming Growth Factor-α (TGF-α) Basic Fibroblast Growth Factorbasic (bFGF), Platelet Derived Promote angiogenesis, also Growth Factors(PDGF) AA, AB and BB, Vascular Endothelial proliferative and migrationstimulatory Growth Factor (VEGF), VEGF-C and VEGF-D effects TGF-β3,Hepatocyte Growth Factor (HGF) Inhibit scar formation α2-macroglobulinInhibit protease activity, coordinate growth factor bioavailabilityAdiponectin (Acrp-30) Regulate growth and activity of keratinocytesGranulocyte Colony-Stimulating Factor (G-CSF) Stimulate stem cellmigration and proliferation Interleukin 1 Receptor Antagonist (IL-1RA)Anti-inflammatory Neutrophil Gelatinase-Associated Lipocalin (N-GAL)Anti-bacterial Leukemia Inhibitory Factor (LIF) Support of angiogenicgrowth factors SDF-1β Recruit cells from circulation to site of tissuedamage Insulin-like Growth Factor Binding Protein (IGFBP1, 2, 3)Regulate IGF and its proliferative effects

Example 11 Cell Phenotype

FACS was performed to determine cell phenotype in a placental product ofthe present invention. Placental products were prepared according to theprocedure detailed in Example 1 through Example 6. The products werethawed and subsequently filtered through a 100 μm filter to removetissue debris. Single cell suspensions were then centrifuged using aBeckman TJ-6 at 2000 rpm for 10 min and washed twice with DPBS.Supernatant was discarded after each wash, and cells were resuspended in2 mL of FACS staining buffer (DPBS+0.09% NaN₃+1% FBS).

Once the single cell suspensions were prepared, a minimum of 1×10⁵ cellsin 100 μL of FACS staining buffer was treated with antibodies labeledwith fluorescent dye. Table 4 provides descriptions of the antibodiesand the amounts used. For cell surface markers, cells were incubated for30 min at room temperature in the dark with antibodies followed bywashing twice with FACS staining buffer by centrifugation at 1300 rpmfor 5 min using a Beckman TJ-6 centrifuge. Cells were then resuspendedin 400 μL of FACS staining buffer and analyzed using a BD FACSCaliburflow cytometer. Results indicate that a placental product derived fromchorion contains live cells which stain positive for MSC markers (FIG.6), implicating the presence of MSC-like cells.

TABLE 4 FACS Antibodies Volume of Cell antibody Cell marker antibodysolution marker Cell marker and label type Cat No. used type specificityIgG1 isotype-PE BD 559320  5 μL Cell Isotype control surface CD105-PECaltag 20 μL Cell MSC marker MHCD10504 surface CD166-PE BD 559263 80 μLCell MSC marker surface CD45-PE BD 555483 10 μL Cell Hematopoeticsurface cell marker

Example 12 Optimization of Cryoprotectants

A placenta was processed according to the procedure detailed in Example1 through Example 2. The resulting digestion suspension comprising cellswas divided into several aliquots, and each processed according to theprocedure detailed in Example 3 through Example 5 using a differentcryoprotectant. Three different cryoprotectants were analyzed for theirability to enhance the number of viable cells recovered after freezingand to preserve protein recovery after freezing. The followingcryoprotectant solutions were tested:

-   -   1. 10% DMSO and 5% HSA in Plasma-Lyte A (CTR solution)    -   2. 5% DMSO and 5% HSA in Plasma-Lyte A    -   3. 10% DMSO in Saline    -   4. 5% DMSO in Saline    -   5. 10% Glycerol in Saline

Before freezing and after thawing, cells were counted using ahemocytometer and trypan blue staining was used to distinguish livecells. The following formula was used to calculate the number of cellsper mL of homogenate: Cells per ml=(# Cells counted per four 0.0001 mLsquares)×10,000×dilution factor. The results are depicted in FIG. 7.

Example 13 Time Course Optimization of Collagenase Digestion ofChorionic Tissue

To determine the optimal time to digest a placental tissue such aschorionic tissues in collagenase II, chorionic tissues from threedifferent donors were analyzed. The tissues were incubated overnight inan antibiotic cocktail. Each chorionic membrane tissue was then washedtwice to remove antibiotic solution and split into three pieces. Eachpiece of tissue was weighed to obtain an initial weight (0 min.) beforebeing digested for 10, 20, or 30 minutes in collagenase II solution (300U/mL).

At the end of each digestion period, the remaining tissue was separatedfrom the collagenase II solution containing the isolated cells byfiltering through a 100 um pore cell filter. The separated tissue wasthen weighed while the collagenase II solution containing digested cellswas centrifuged. The resulting cell pellet was resuspended in PBS andcounted using a hemocytometer with trypan blue exclusion.

The weight of each remaining tissue piece, including the weight oftissue remaining on the cell filter, was used to calculate the percentof weight lost by digestion with collagenase II.

As shown in FIG. 8, after 10 min. of digestion, about 10% of theoriginal tissue weight was reduced. Further incubation resulted in amore dramatic loss of weight. By 30 minutes, nearly half of the originalweight was lost. It was further noted that tissue digested for longerthan 10 min. became extremely difficult to separate from the collagenaseII solution.

FIG. 8 also shows the number of cells released by collagenase digestion.After 10 minutes of incubation, a substantial number of cells werereleased. However, by 20 minutes, the number of cells released increasedby about 4-fold.

These results surprising demonstrate that by performing only a limitedcollagenase digestion (e.g. about 10 minutes), a substantial number ofplacental cells can be released and the integrity of the placentaltissue is maintained. Accordingly, when the limited collagenase digestedplacental tissue is subsequently disrupted, the dispersion retains asubstantial amount of its native character. For example, the inventorsgenerally observe that after prolonged collagenase digestion (e.g. 30minutes), the placental tissue can be passed through a 100 micronfilter. This is in contrast to the limited digestion where substantiallyless (e.g. one half or one quarter or less) of the tissue can be passedthrough a 100 micron filter.

When this dispersion is combined with the released placental cells, asuperior therapeutic product is produced.

In data not shown, no significant change in the viability of thecollagenase-released cells was observed through 30 min. of digestion.

Example 14 Time Course Optimization of Collagenase Digestion of AmnioticTissue

The limited digestion method of Example 13 was tested for applicabilitywhen the placental tissue is amniotic tissue. The following procedurewas performed:

-   -   1. Process placenta.        -   a. Remove amniotic tissue and wash twice in PBS.        -   b. Soak amniotic tissue to loosen red blood cells.            -   i. If needed, clear red blood cells from tissue using                fingers.        -   c. Incubate amniotic tissue for 24 hrs. in antibiotic            cocktail.    -   2. Remove amniotic tissue from antibiotic cocktail and wash        twice in PBS.    -   3. Incubate amniotic tissue for 30 min at 37° C. in 200 mL        trypsin solution (0.25%).    -   4. Remove amniotic tissue from trypsin solution and wash twice        in PBS.    -   5. Incubate amniotic tissue for 10 min at 37° C. in 200 mL        collagenase II solution (300 U/mL in DMEM).    -   6. Remove amniotic tissue from collagenase II solution and wash        twice in PBS.    -   7. Processing of collagenase II and trypsin live cell        suspensions.        -   a. Centrifuge each suspension at 2000 rcf for 5 min.        -   b. Pour off each supernatant and replace with 10 mL PBS.            -   i. Resuspend cells in PBS to wash.        -   c. Centrifuge cell suspension at 2000 rcf for 5 min.        -   d. Pour off supernatants and resuspend cells in 2 mL            cryprotectant (5% DMSO in saline).        -   e. Combine pellets.    -   8. Processing of amniotic tissue.        -   a. Place amniotic tissue in homogenization container with a            volume of cryoprotectant (mL) equal to the weight of the            amniotic membrane (g). For example, if the amniotic membrane            weight 25 g place it in the homogenization container with 25            mL of cryoprotectant.        -   b. Allow the amniotic tissue and cryoprotectant to sit on            ice for at least 10 min.        -   c. Homogenize at high speed twice for 5 sec. using a tissue            homogenizer.    -   9. Combine isolated live cells with homogenate and mix        thoroughly (the “placental product”).    -   10. Aliquot into vials and place at 4° C. for 30-60 min.        -   Freeze at −80° C. until use.

To determine the mean number of live cells in the amnion homogenate,multiple placentas were prepared. Each amnion was processed in onepiece, and cell counts were obtained post thaw after cryopreservation(incubation at 4° C. and subsequent freezing at −80° C.). All cell countdata were pooled, and a mean was calculated.

Samples from each donor were also prepared for protein array analysis.Briefly, 1 mL of homogenate from each donor was centrifuged at 14,000rpm in a microcentrifuge for 10 min. The resulting supernatant from eachsample was collected. Supernatants along with positive and negativecontrols were sent to Aushon Biosystems for analysis using theirSearchLight protein array assay. This assay measures the levels of 37proteins of interest in each sample. For this experiment, negativecontrol samples consisted of 5% DMSO in saline (cryopreservationsolution), and positive control samples consisted of cryopreservationsolution with known concentrations of spiked recombinant proteins (bFGF,EGF, and VEGF).

FACS analysis of single cell suspensions from the placental product wasperformed for the markers CD45, CD 105, and CD 166.

Results.

As shown in FIG. 9, limited collagenase digestion of amniotic membranetissue resulted in release of a substantial number of live placentalcells.

As shown in Table 5, limited collagenase digestion of amniotic membranetissue preserved placental factors in the placental dispersion madetherefrom.

When Example 13 and Example 14 are considered together, it is nowconcluded that limited collagenase digestion of placental tissue,whether it be chorion tissue, amniotic tissue, or other tissue ofplacental origin, results unexpectedly in:

Substantial numbers of release live placental cells;

Preserved endogenous placental factors;

Preserved endogenous placental protein (e.g. matrix proteins); and

A therapeutically effective product.

TABLE 5 Therapeutic Profiles of Amnion-Derived Placental Products Max.Mean Protein Min. (pg/mL) (pg/mL) (pg/mL) MMP1 6697.73 10010.27 8354MMP2 5456.52 53432.45 29444.49 MMP3 570.97 579.1 575.04 MMP7 74.11207.31 140.71 MMP8 3829.63 3978.42 3904.03 MMP9 11735.19 43661.6327698.41 MMP10 38916.81 51526.9 45221.86 MMP13 TLTD TLTD TLTD TIMP131427.94 78147 54787.47 TIMP2 6149.25 23167.29 14658.27 TSP1 TLTD TLTDTLTD TSP2 7741.98 13312.64 10527.31 TGFα TLTD TLTD TLTD TGFβ1 85.17350.51 217.84 TGFβ2 47.98 58.6 53.29 bFGF (FGF-2) 19305.72 23427.4821366.6 KGF (FGF-7) 70.39 94.29 82.34 EGF 13.71 69.55 41.63 HB-EGF TLTDTLTD TLTD PDGFAA 14.47 27.93 21.2 PDGFAB TLTD TLTD TLTD PDGFBB 7.4912.34 9.91 VEGF 346.3 1058.85 702.57 VEGFC 114.35 220.27 167.31 VEGFD49.54 75.29 62.42 HGF 12068.53 17408.53 14738.53 PEDF TLTD TLTD TLTDANG2 TLTD TLTD TLTD IGFBP1 128.6 159.84 144.22 IGFBP2 TLTD TLTD TLTDIGFBP3 699.01 1349.06 1024.04 ACRP30 6677.35 11232.13 8954.74Fibronectin 141595.2 254184.05 197889.63 Alpha2mac 421402.95 790851606126.98 IL1ra 7542.74 10535.55 9039.14 NGAL 1521.63 3283.59 2402.61SDF1b TLTD TLTD TLTD TLTD = too low to detect

Example 15 Live Cells from the Placental Dispersions and the PlacentalCell Components of the Placental Product

The manufacturing steps taught here (e.g. limited collagenase digestion,removal of placental cells before placental tissue disruption, andlimited disruption methods) result in a highly effective therapeuticproduct. The relative role of the placental dispersion and the placentalcells components were evaluated for respective role in providing livecells.

Chorionic tissue was obtained from placental tissue of 9 subjects andthe placental cells (e.g. collagenase-released) and placental dispersionwas assessed for the number of live cells.

TABLE 6 Placental Cells from Placental Cell and Placental DispersionFractions Cells in the Placental Cells in Theoretical cell fraction thePlacental cells in the Donor (collagenase-released) dispersion fractionplacental product D144 3.84E+05 7.95E+06 8.33E+06 D145 8.40E+05 1.25E+071.33E+07 D146 1.60E+05 7.84E+06 8.00E+06 D147 2.17E+07 5.70E+06 2.74E+07D153 3.26E+06 1.64E+07 1.97E+07 D154 3.70E+05 1.07E+07 1.11E+07 D1552.08E+06 7.10E+06 9.18E+06 D156 4.90E+05 1.26E+07 1.31E+07 Mean 3.66E+061.01E+07 1.38E+07

As shown in Table 6, 21% to 98% of the cells in the placental productswere derived from the placental dispersion component. Thus, the methodsof the present invention unexpectedly preserve important placentalfactors and live cells in the placental dispersion and also providesubstantial numbers of live cells from the placental cell(collagenase-released) component.

Example 16 Hypoxia Treatment

Results from private studies indicate that hypoxia induces many proteinshaving beneficial functions in the process of burn wound healing.However, the extent to which hypoxia effects cell growth and proteinexpression depends on the specific conditions of its application.Therefore, several experiments were performed to determine if hypoxiacould enhance the effectiveness of chorion-derived placental products.

A placenta was processed according to the procedure detailed in Example1, except the chorionic membrane was divided into two halves beforetreatment with the antibiotic cocktail. One half of the chorionicmembrane tissue was incubated under hypoxic conditions (1% O2) while theother was incubated under normal cell culture conditions (˜20% O2). Eachhalf was then process as described in Example 2 through Example 5.Before freezing and after thawing, cells were counted using ahemocytometer and trypan blue staining was used to distinguish livecells. The results are depicted in FIG. 10.

Example 17 Hypoxia Treatment and Cryoprotectants

A placenta was processed according to the procedure detailed in Example15 except that the digests from each half of the chorionic membrane werefurther split and formulated with different cryoprotectants, as inExample 12. Before freezing and after thawing, cells were counted usinga hemocytometer and trypan blue staining was used to distinguish livecells. The data are presented in FIG. 11. As depicted in FIG. 11,processing under normoxic conditions provides superior cell viability.Also as depicted in FIG. 11, subjecting the chorion to hypoxicconditions may be detrimental to cell viability.

Example 18 Growth Factors are Expressed for a Minimum of 14 days

Placental products of the present invention demonstrate a durableeffect, desirable for wound healing treatments. The extracellular matrixand presence of viable cells within the placental product derived fromthe chorionic membrane described in this invention allow for a cocktailof proteins that are known to be important for wound healing to bepresent for at least 14 days.

Placental product derived from the chorionic membrane were thawed andplated onto tissue culture wells and incubated at 37° C.±2° C. for 3, 7,and 14 days. At each time point, a sample of the product was collectedand centrifuged at 16,000 g for 10 min to collect the supernatant. Thesupernatants were then run on ELISAs for bFGF and VEGF. FIG. 12illustrates the duration of two key wound healing proteins, bFGF andVEGF, at 3, 7 and 14 days. Although the expression of bFGF goes downwith time, it should be noted that significant levels of bFGF waspresent even out to 14 days. Interestingly, the expression of VEGFincreased with time, which could be due to continued active expressionof VEGF from the viable cells within the placental product derived fromthe chorionic membrane.

Example 19 Interferon 2α (IFN-2α) and Transforming Growth Factor-β3(TGF-β3)

Interferon-2α and TGF-β3 have been described in the literature asplaying critical roles in the prevention of scar and contractureformation (Kwan et al., Hand Clin, 2009, 25:511; Tredget et al., SurgClin North Am 1997, 77:701). IFN-2α is known to decrease collagen andfibronectin synthesis and fibroblast-mediated wound contracture.Clinically, IFN-2α has been administered subcutaneously and shown toimprove scar quality (Nedelec et al, Lab Clin Med 1995, 126:474). TGF-β3regulates the deposition of extracellular matrix and has been shown todecrease scar formation when injected in rodent cutaneous wound models.Clinically, TGF-β3 has been shown to improve scar appearance wheninjected at the wound site (Occleston et al., J Biomater Sci Polym Ed2008, 19:1047). Placental product derived from the chorionic membranedescribed in this invention has been analyzed for the presence of IFN-2αand TGF-β3. Briefly, placental product derived from the chorionicmembrane was thawed and centrifuged at 16,000 g to collect supernatants.Supernatants were analyzed on a commercially available ELISA kit fromMabTech (IFN-2α) and R&D Systems (TGF-β3). FIG. 13 shows significantexpression of IFN-2α and TGF-β3 in placenta products derived from thechorionic membrane.

Example 20 Tissue Reparative Proteins in Chorionic Membrane Homogenates

Placental product derived from the chorionic membrane was analyzed forthe presence of proteins that are important in tissue repair.

Placental products derived from chorionic membranes described in thisinvention were analyzed for the presence of these tissue reparativeproteins. Briefly, placental product derived from the chorionic membranewas incubated at 37° C.±2° C. for 72 hrs. The product was centrifuged,and the supernatant was analyzed on commercially available ELISA kitsfrom R&D Systems. FIG. 14 shows significant expression of BMP-2, BMP-4,BMP-7, PLAB, PIGF, and IGF-1 in several donors of placental productsderived from chorionic membranes.

Without being bound by theory, the inventors believe that efficacy ofthe present placental products for wound repair are due, in part, to therole of BMPs, IGF-1, and PIGF in the development and homeostasis ofvarious tissues by regulating key cellular processes. BMP-2 and BMP-4may stimulate differentiation of MSCs to osteoblasts in addition topromote cell growth; placental BMP or PLAB is a novel member of the BMPfamily that is suggested to mediate embryonic development. Insulin-likegrowth factor 1 (IGF-1) may promotes proliferation and differentiationof osteoprogenitor cells. Placental derived growth factor (PIGF) mayacts as a mitogen for osteoblasts.

Without being bound by theory, the inventors believe that efficacy ofthe present placental products for wound repair are due, in part, to therole of BMPs, IGF-1, and PIGF in the development and homeostasis ofvarious tissues by regulating key cellular processes. BMP-2 and BMP-4may stimulate differentiation of MSCs to osteoblasts in addition topromote cell growth; placental BMP or PLAB is a novel member of the BMPfamily that is suggested to mediate embryonic development. Insulin-likegrowth factor 1 (IGF-1) may promotes proliferation and differentiationof osteoprogenitor cells. Placental derived growth factor (PIGF) mayacts as a mitogen for osteoblasts.

Example 21 Differentiation Capacity of Cells Derived from the ChorionicMembrane

Placental cells, in optional embodiments of the present invention, areadherent, express specific cellular markers such as CD105 and lackexpression of other markers such as CD45, and demonstrate the ability todifferentiate into adipocytes, osteoblasts, and chondroblasts.

The expression of specific cellular markers has already been describedin Example 20. To determine if the cells within the placental productderived from the chorionic membrane can adhere to plastic anddifferentiate into one of the lineages, cells were isolated from theplacental product derived from the chorion as described in thisinvention and cultured at 37° C.±2° C. and expanded.

FIG. 15-A shows a representative image of passage 2 cells, demonstratingthe ability of the cells to adhere to tissue culture plastic. As acomparison, a representative image of MSCs isolated and expanded fromhuman bone marrow aspirate is shown in FIG. 15-B.

Osteogenic differentiation capacity was demonstrated by staining thecultured cells with alkaline phosphatase labeling following themanufacturer's recommendations (BCIP/NBT Alkaline Phosphatase SubstrateKit IV, Vector Laboratories Cat. No. SK-5400). Alkaline phosphatase isan enzyme involved in bone mineralization (Allori et al., TissueEngineering: Part B, 2008, 8:275), and its expression within cells isindicative of osteo-precursor cells (Majors et al., J Orthopaedic Res,1997, 15:546). Staining for alkaline phosphatase is carried out throughan enzymatic reaction with Bromo-4-Chloro-3′-Indolylphosphatep-Toluidine Salt (BCIP) and Nitro-Blue Tetrazolium Chloride (NTP). BCIPis hydrolyzed by alkaline phosphatase to form an intermediate thatundergoes dimerization to produce an indigo dye. The NBT is reduced tothe NBT-formazan by the two reducing equivalents generated by thedimerization. Together these reactions produce an intense, insolubleblack-purple precipitate when reacted with alkaline phosphatase.

FIG. 15-C shows a representative image of passage 2 cells isolated andexpanded from placental product derived from the chorionic membranestaining positively for alkaline phosphatase.

1-36. (canceled)
 37. A method of manufacturing a therapeutic placentalproduct comprising: a) obtaining a first placental tissue; b) obtainingplacental cells from the first placental tissue; c) obtaining a secondplacental tissue; d) disrupting the second placental tissue to form adispersion comprising placental factors; and e) combining the placentalcells and the placental dispersion to form the placental product,wherein the placental dispersion is a homogenate.
 38. The method ofclaim 37, wherein the first placental tissue is autologous to the secondplacental tissue and wherein the first placental tissue and the secondplacental tissue are chorionic tissue.
 39. The method of claim 38,wherein the second placental tissue is derived from the fist placentaltissue after the step of obtaining the placental cells.
 40. The methodof claim 39, wherein the step of obtaining the placental cells comprisesexposing the first placental tissue to a protease, optionally whereinthe protease is a collagenase.
 41. The method of claim 40, wherein theprotease exposing step has a duration of about 30 minutes or less. 42.The method of claim 40, wherein the protease exposing step releases lessthan about 10% of the maximum number of releasable placental cells. 43.The method of claim 38, wherein the chorionic tissue is depleted oftrophoblasts.
 44. The method of claim 43, wherein the trophoblasts aredepleted by dissection and dispase treatment.
 45. The method of claim37, wherein the placental product comprises one or more members selectedfrom the group consisting of extracellular matrix proteins; proteaseinhibitors; angiogenic factors; and placental factors which promotes themigration of epithelial cells into a wound.
 46. The method of claim 37,wherein the placental product comprises one or more protease inhibitorsselected from the group consisting of matrix metalloproteinases (TIMPs),alpha-2 macroglobulin, and thrombospondins.
 47. The method of claim 37,wherein the placental product comprises one or more angiogenic factorsselected from the group consisting of VEGF and bFGF.
 48. The method ofclaim 37, wherein the placental product comprises one or more factorswhich promote the migration of epithelial cells into a wound selectedfrom the group consisting of HGF and KGF.
 49. The method of claim 37,wherein the placental cells are selected from the group consisting ofMSCs, ESCs, placenta-derived mesenchymal progenitor cells, placentalmesenchymal stem cells fibroblasts, epithelial cells, placentalmesenchymal cells, and macrophages.
 50. The method of claim 49, whereinthe placental cells are present at a concentration of at least about20,000 per ml of placental product.
 51. The method of claim 37, whereinat least one of the placental cells or the placental dispersion iscryopreserved.
 52. The method of claim 37, wherein the placental cellsare cryopreserved before combining with the placental dispersion, andwherein the placental dispersion is optionally cryopreserved beforecombining with the placental cells.
 53. The method of claim 37, whereinthe placental cells comprise stromal cells and the placental cells arecryopreserved.
 54. The method of claim 39, wherein: a. the step ofobtaining the placental cells comprises exposing the first placentaltissue to a protease; b. the protease exposing step is of about 30minutes or less duration; c. the protease exposing step releases lessthan about 10% of the maximum number of releasable placental cells; d.the chorionic tissue is depleted of trophoblasts; e. the placentalproduct comprises one or more protease inhibitors selected from thegroup consisting of matrix metalloproteinases (TIMPs), alpha-2macroglobulin, and thrombospondins; f. placental product comprises oneor more of VEGF and bFGF; g. the placental product comprises one or moreof HGF and KGF; h. the placental cells comprise MSCs and cells selectedfrom the group consisting of ESCs, placenta-derived mesenchymalprogenitor cells, placental mesenchymal stem cells fibroblasts,epithelial cells, placental mesenchymal cells, and macrophages; i. theplacental cells are present at a concentration of at least about 20,000per ml of placental product; and j. at least one of the placental cellsand the placental dispersion is cryopreserved.
 55. A method of treatinga tissue injury comprising administering to a subject in need thereof atherapeutic placental product made by the method of claim 37.